Polyglutamine (PolyQ) diseases are progressive neurodegenerative disorders caused by both protein- and RNA-mediated toxicities. We previously showed that a peptidyl inhibitor, P3, which binds directly to expanded CAG RNA can inhibit RNA-induced nucleolar stress and suppress RNA-induced neurotoxicity. Here we report a N-acetylated and C-amidated derivative of P3, P3V8, that showed a more than 20-fold increase in its affinity for expanded CAG RNA. The P3V8 peptide also more potently alleviated expanded RNA-induced cytotoxicity in vitro, and suppressed polyQ neurodegeneration in Drosophila with no observed toxic effects. Further N-palmitoylation of P3V8 (L1P3V8) not only significantly improved its cellular uptake and stability, but also facilitated its systemic exposure and brain uptake in rats via intranasal administration. Our findings demonstrate that concomitant N-acetylation, C-amidation and palmitoylation of P3 significantly improve both its bioactivity and pharmacological profile. L1P3V8 possesses drug/lead-like properties that can be further developed into a lead inhibitor for the treatment of polyQ diseases.

DNA nanostructures have been designed and used in many differenta pplications.H owever,t he use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimicsi so nly starting to emerge. Herein five coiledcoil peptide structures were templatedb yt he hybridization of a d-DNA triplex or its mirror-image counterpart, an l-DNA triplex.T he self-assembly of the desiredt rimeric structures in solutionw as confirmed by gel electrophoresis and smallangle X-ray scattering,a nd the stabilizing synergyb etween the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l-DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-basedn anotechnology field by adding two extra dimensions, that is, chirality and polarity,t op rovide innovative molecular tools forr ational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.[a] Dr.[a] Thermal denaturation and annealing temperatures (T m and T a values) of POC and ON-reference triple helixes measured at pH 5.5 as an average of three independent melting temperature determinations shownw ith the corresponding standard deviations. The values in brackets are T m and T a values measured for the corresponding underlying duplexes. The experiments werer ecorded at 275 nm in 10 mm acetate buffer( NaOAc/HOAc)c ontaining1 00 mm NaCl. The concentration of the individual duplex components was 1.0 mm while the TFO component was used in 1.5 mm concentration. The peptide moiety is marked in yellowa nd red (l-peptidea sr ight-handed helix, d-peptidea sl eft-handedh elix), the TFO moiety in dark blue (d-nucleotide) or light blue (l-nucleotide),a nd the DNA duplexm oiety in crimson (d-nucleotide) or in orange (l-nucleotide).

Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein.To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 μmol/kg) and L1P3V8 (6 μmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA-and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their braintargeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model.